Lipidated glycosaminoglycan particles and their use in drug and gene delivery for diagnosis and therapy

ABSTRACT

Lipidated glycosaminoglycan particles, prepared by reacting a glycosaminoglycan with at least one lipid to cross-link the carboxylic acid groups in the glycosaminoglycan with a primary amine in the lipid, are used to encapsulate drugs for use in the treatment of pathological conditions in an animal.

FIELD OF THE INVENTION

The present invention is directed to a drug delivery system based uponparticles of lipidated glycosaminoglycans which encapsulate drugs forsubsequent delivery for use in therapy and diagnosis.

BACKGROUND OF THE INVENTION

Glycosaminoglycans, or mucopolysaccharides, along with collagen, are thechief structural elements of all connective tissues. Glycosaminoglycans,or gags, are large complexes of polysaccharide chains associated with asmall amount of protein. These compounds have the ability to bind largeamounts of water, thereby producing a gel-like matrix that forms thebody's connective tissues. Gags are long chains composed of repeatingdisaccharide units (aminosugar-acidic sugar repeating units). Theaminosugar is typically glucosamine or galactosamine. The aminosugar canalso be sulfated. The acidic sugar may be D-glucuronic acid orL-iduronic acid. In vivo, gags other than hyaluronic acid are covalentlybound to a protein, forming proteoglycan monomers. The polysaccharidechains are elongated by the sequential addition of acidic sugars andaminosugars.

Among the most common gags are hyaluronic acid, keratan sulfate,chondroitin sulfate, heparin sulfate, and dermatin sulfate. Gags may bechemically modified to contain more sulfur groups than in theirinitially extracted form. In addition, gags may be partially orcompletely synthesized and may be of either plant or animal origin.

Hyaluronic acid is a naturally occurring member of the glycosaminoglycanfamily which is present in particularly high concentration in thecartilage and synovial fluid of articular joints, as well as in vitreoushumor, in blood vessel walls, and umbilical cord and other connectivetissues. Hyaluronic acid can be in a free form, such as in synovialfluid, and in an attached form, such as an extracellular matrixcomponent. This polysaccharide consists of alternatingN-acetyl-D-glucosamine and D-glucuronic acid residues joined byalternating beta-1,3-glucuronidic and beta-1,4-glucosaminidic bonds. Inwater, hyaluronic acid dissolves to form a highly viscous fluid. Themolecular weight of hyaluronic acid isolated from natural sourcesgenerally falls within the range of 5×10⁴ up to 10⁷ daltons. Hyaluronicacid has a high affinity for the extracellular matrix and to a varietyof tumors, including those of the breast, brain, lung, skin, and otherorgans and tissues.

A drug delivery system is used for maintaining a constant blood level ofa drug over a long period of time by administering a drug into the body,or for maintaining an optimal concentration of a drug in a specifictarget organ by systemic or local administration, and over a prolongedperiod of time.

Chemically modified hyaluronic acid can be used for controlled releasedrug delivery. Balazs et al, in U.S. Pat. No. 4,582,865, state that“cross-linked gels of hyaluronic acid can slow down the release of a lowmolecular weight substance dispersed therein but not covalently attachedto the gel macromolecular matrix.”

Various forms of pharmaceutical preparations are used as drug deliverysystems, including the use of a thin membrane of a polymer or the use ofa liposome as a carrier for a drug.

There are two basic classes of drug carriers: particulate systems, suchas cells, microspheres, viral envelopes, and liposomes; andnon-particulate systems, which are usually soluble systems, consistingof macromolecules such as proteins or synthetic polymers.

Generally, microscopic and submicroscopic particulate carriers haveseveral distinct advantages. They can perform as sustained-release orcontrolled-release drug depots, thus contributing to improvement in drugefficacy and allowing reduction in the frequency of dosing. By providingprotection of both the entrapped drug and the biological environment,these carriers reduce the risks of drug inactivation and drugdegradation. Since the pharmacokinetics of free drug release from theparticles are different from directly-administered free drug, thesecarriers can be used to reduce toxicity and undesirable side effects.

Despite the advantages offered, there are some difficulties associatedwith using drug encapsulating biopolymers. For example, biopolymersstructured as microparticulates or nanoparticulates have limitedtargeting abilities, limited retention and stability in circulation,potential toxicity upon chronic administration, and the inability toextravasate. Numerous attempts have been made to bind differentrecognizing substances, including antibodies, glycoproteins, andlectins, to particulate systems, such as liposomes, microspheres, andothers, in order to confer upon them some measure of targeting. Althoughbonding of these recognizing agents to the particulate system has metwith success, the resulting modified particulate systems did not performas hoped, particularly in vivo.

Other difficulties have also arisen when using such recognizingsubstances. For example, antibodies can be patient-specific, and therebyadd cost to the drug therapy. Additionally, not all binding betweenrecognizing substrate and carrier is covalent. Covalent bonding isessential, as non-covalent binding might result in dissociation of therecognizing substances from the particulate system at the site ofadministration, due to competition between the particulate system andthe recognition counterparts to the target site for the recognizingsubstance. Upon such dissociation, the administered modified particulatesystem can revert to a regular particulate system, thereby defeating thepurpose of administration of the modified particulate system.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the deficiencies inthe prior art.

It is another object of the present invention to formglycosaminoglycan-based particles for encapsulating drugs.

It is another object of the present invention to deliver drugsencapsulated in a glycosaminoglycan-based particle.

It is a further object of the present invention to provide methods ofdrug delivery using particles of lipidated glycosaminoglycans as thedrug delivery vehicles.

In a preferred embodiment, the delivery is by oral administration of theparticle formulation. In another preferred embodiment, the delivery isby intranasal administration of the particle formulation, especially foruse in therapy of the brain and related organs (e.g., meninges andspinal cord) that seeks to bypass the blood-brain barrier (BBB). Alongthese lines, intraocular administration is also possible. In anotherpreferred embodiment, the delivery means is by intravenous (i.v.)administration of the particle formulation, which is especiallyadvantageous when a longer-lasting i.v. formulation is desired.

It is still another object of the present invention to provide genedelivery using particles of lipidated glycosaminoglycans as the genedelivery materials.

The present invention provides a novel multi-product gene and drugdelivery technology as well as methods of preparation and uses thereof.The delivery system comprises lipidated glycosaminoglycans, also knownas gagomers, which are bioadhesive biopolymers produced by cross-linkinga lipid having a primary amino group to a carboxylic acid-containingglycosaminoglycan. Micro- or nanoparticles are formed in a controlledmanner, with dominant particle diameter ranges of about 2-5 microns formicroparticles and about 50-200 nanometers for nanoparticles. Eithersmall or large drugs, bioactive agents, or active ingredients such asantibiotics, chemotherapeutics, proteins, and nucleic acids can beentrapped in these particles with high efficiency, usually greater than50%, even for large macromolecules. For example, for plasmid DNA thenanoparticles provide about 66% entrapment and the microparticlesprovide about 75% entrapment.

For purposes of the present invention, “drug” means any agent which canaffect the body therapeutically, or which can be used in vivo fordiagnosis. Examples of therapeutic drugs include chemotherapeutics forcancer treatment, antibiotics for treating infections, and antifungalsfor treating fungal infections. Examples of diagnostic drugs includeradioactive isotopes such as ⁹⁹Tc, ¹²⁷I, and ⁶⁷Gd, and fluorescentmolecules which are used in visualizing sites of interest in the body.

Preparation of the biopolymers of the present invention and drugentrapment are simple and cost-effective processes. These novel carriersact as sustained release drug depots, with half-lives in the range of19-35 hours for the efflux of antibiotics and chemotherapeutics. Theseproperties, together with their bioadhesive nature, provide these noveldrug carriers the ability to perform as site-adherent, site-retained,sustained release drug depots for systemic, including oral, topical, andregional, including intranasal, administrations.

Additionally, the gagomers of the present invention are non-toxic. Whenchemotherapeutic drugs were entrapped and tested in a cell culturemodel, the systems exhibited high potential in tumor treatment, evenovercoming the well known impediment of drug resistance. Thus, thegagomers can be used as microscopic and submicroscopic drug deliverysystems for a wide range of therapeutic activities, such as cancer,infectious diseases, wound healing, enzyme therapy, gene therapy, andothers.

Unexpectedly, the empty particles (containing only gagomers and no drugor other therapeutic formulation) also appear to have importanttumor-inhibiting effects. Therefore, such particles may be useful forcancer therapy, especially for metastic cancer, either as a main oradjuvant chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are scanning electron microscopy pictures of fields ofparticles from the same batch at two different magnifications. FIG. 1Ais at 5000× magnification. FIG. 1B is at 3000× magnification.

FIGS. 2A-2C are confocal micrographs showing individual cells incubatedwith three different formulations. FIG. 2A shows cells of the C6 (ratglioma) line that were incubated with a free ethidium bromide (EtBr).FIG. 2B shows cells of the C6 that were incubated with “empty” (i.e.,entrapping buffer alone) gagomers suspended in a solution of free EtBr.FIG. 2C shows cells of the C6 that were incubated with EtBr-entrappinggagomers.

FIG. 3A shows cells of the PANC-1 cell line (from human pancreaticadenocarcinoma) treated with gagomer-encapsulated EtBr.

FIG. 3B shows cells of the PANC-1 cell line treated with free EtBr.

FIG. 4A is a confocal micrograph of a system similar to FIG. 3B, but ata larger magnification.

FIG. 4B is a confocal micrograph of a system similar to FIG. 3A, but ata larger magnification.

FIG. 5 shows the results of turbidity studies of free hyaluronic acidand a hyaluronic acid-based gagomer as a function of macromolecularconcentration, following absorbency changes at 600 nm in the form of agraph plotting concentration of free hyaluronic acid and a hyaluronicacid-based gagomer.

FIGS. 6A and 6B shows microscopy of microgagomers and nanogagomers. FIG.6A shows fluorescent microscopy of micro-gagomers entrapping a modelprotein, BSA-FITC, magnification factor: 2000. FIG. 6B shows lightmicroscopy of nanogagomers entrapping plasmid DNA, magnification factor:2000.

FIG. 7 is a graph illustrating doxorubicin efflux from micro (round) andnano (square) gagomers under conditions of unidirectional flux. Theindependent variable is time. The dependent variable (f) is thepercentage of drug released at time=t with respect to the total drug inthe system at time t=0. The symbols represent the experimental data andthe solid curves are the theoretical expectations according to amulti-pool efflux mechanism.

FIG. 8 is a graph showing survival of C6 cells 48 hours post-treatmentby free micro-gagomer (i.e., encapsulating buffer alone, as in “empty”defined in the description to FIG. 2B), a given dose of a freechemotherapeutic drug, and an equivalent dose of the same drug entrappedin the micro-gagomer. The studies were conducted with mitomycin c (MMC),doxorubicin (DOX), and vinblastine (VIN), and the results are organizedinto three data sets, one for each drug. Each bar is an average of threeindependent experiments, each of which comprised 20 separatemeasurements. The error bars represent the respective standarddeviations.

FIG. 9 shows the zeta potentials (effective surface charge) of the nano-and microparticles as a function of concentration. Zeta potentialsreflect the total interaction forces between colloidal size particles insuspension.

FIG. 10 depicts the results of toxicity testing of free drug deliverysystem (DDS) nanoparticles. DDS dose was 1 mg/ml and the incubation timewas 24 hours. Each bar is an average of 32-64 independentdeterminations, and the error bars represent the standard deviations.

FIGS. 11A and 11B show the cytotoxic effects of MMC (FIG. 11A) and ofDOX (FIG. 11B), formulated in the DDS (nano particles) in C26 cellsexposed to the treatment media for 4 hours, compared to free drug andfree drug delivery system (DDS). *** indicates p<0.001, comparing foreach drug species and dose the carrier vs. free formulations.

FIG. 12 depicts the concentrations of MMC in blood, as a function offormulation type and time from injection. Each symbol is an average of 5animals and the error bars represent the standard error of the mean(SEM). The lines are non-theoretical, drawn to emphasize the trends ofthe data.

FIG. 13 illustrates the increase in tumor volume with time. Points areexperimental, each an average of 5 animals; the error bars are the SEMand the curves are non-theoretical, indicating the trends in the data.The arrows and the numbers above them indicate treatment days. Thenumbers next to the symbols are days of tumor appearance.

FIG. 14 illustrates the survival of the animals in Run 1. Each animalreceived 3 injections of the selected formulation. Data for the salineand free MMC groups are from 10 animals/group; data for the free DDS andthe MMS/DDS are from 5 animals/group.

FIG. 15 illustrates the survival of animals in Run 2. Each animalreceived 4 injections of the selected formulation. Data for the free DDSis from 3 animals and for the MMC/DDS from 5 animals.

FIG. 16 is a bar graph showing the brain accumulation of MMC, used as amarker, following intranasal (IN) administration of free MMC and MMCentrapped in DDS nanoparticles. Data is from Run 1, an experiment withrats.

FIG. 17 is a bar graph showing the brain accumulation of MMC, used as amarker, following intranasal (IN) administration of free MMC and MMCentrapped in DDS nanoparticles. Data is from Run 2, an experiment withmice.

FIG. 18 shows the number of metastases found in the lungs of the C57BL/6mice injected i.v. with B16F10 cells. The control group representshealthy animals that were not injected with tumor cells. Each bar is anaverage of the five animals in the group and the error bar is standarddeviation.

FIG. 19 shows the increase in lung weight of tumor-injected micecalculated from the raw data of lungs' weight, according to the formulalisted in the experimental section. Each bar is an average for the 5animals in the group and the error bars are the standard deviation.

FIG. 20 represents a replotting of the data of FIGS. 18 and 19 (averagesonly). The points are the experimental data, and the solid lines arenon-theoretical, drawn to emphasize the trends.

FIG. 21 depicts uptake of BSA-FITC entrapped gagomers into MCF7 cellsusing light and fluorescent microscopy. The upper two panels show uptakeof free protein and non-specific binding. The lower two panels depictprotein entry into the cytosol and nucleus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the preparation and uses of microscopicand submicroscopic delivery systems, as well as materials that can beused for tissue engineering and tissue scaffolding. The drug deliverysystems of the present invention are novel adhesive biopolymers whichtake the form of a particulate carrier, also referred to as a gagomer,made from a lipid which contains at least one primary amine and aglycosaminoglycan, i.e., lipidated glycosaminoglycans.

The particles of the present invention are particularly cost-effectivewhen compared to other particulate carriers, as shown in Table 1.

As used in the present application, the term hyaluronic acid, or HA,refers to hyaluronic acid and any of its hyaluronate salts, including,for example, sodium hyaluronate, potassium hyaluronate, magnesiumhyaluronate, and calcium hyaluronate. Similarly, for any of theglycosaminoglycans, salts as well as free acids are included in the termglycosaminoglycan.

The gagomers of the present invention are microparticulate andnanoparticulate drug delivery systems, also referred to as MDDS andNDDS, respectively, that use drug-entrapping adhesive biopolymers. Thesecarriers, when loaded with drugs, improve clinical outcomes compared tothe same drugs administered in their free form. The gagomers are madefrom naturally-occurring materials which are bio-compatible andbiodegradable.

TABLE 1 Advantages of Present Invention: Aspects of Cost-EffectiveProduction Gagomers Other Particulate Carriers Cost-EffectiveProduction: Raw Materials Stable, available, relatively Some or allcomponents have inexpensive, fit a wide stability and availabilitypatient populations limitations, some may fit only narrow patientpopulations Cost-effective Production: Manufacturing Manufacturingmethodologies Most cases require large used for R & D are amenable toinvestment in developing scale-up with little or no scaled-up productionmethods modifications Cost-effective Production: Production Lines Sinceproduction of the Particle production and drug lipidated-GAG and drugentrapment are done, for entrapment are separate most, in the sameprocess, processes, a single production requiring a separate productline of the lipidated GAG fits line for each final all products. Twopopulations formulation. Likewise for of particle sizes can be differentparticle sizes. fractionated by a simple procedure, from the same batch.Cost-Effective Production: Preparation of Formulation for Use Finalformulation is by simple The final formulation, rehydration of thelipidated- including the entrapped drug, GAG dry powder in an aqueoushas to be provided by the solution of the desired drug. manufacturer.Can be done at patient's bed- side, home, etc. Cost-effectiveProduction: Stability and Shelf Life Drug and lipidated-GAG can beStorage is of the final stored separately in dry form, formulation,namely drug- until reconstitution for use, loaded carrier. Dry form isproviding high stability and not available in all cases. long-term shelflife As a result there are limitations on both stability and shelf life

The gagomers of the present invention have a number of other advantagesover other particulate carriers, including aspects of their in vivofate, as shown in Table 2.

TABLE 2 Advantages of Present Invention - Aspects of In Vivo FateGagomers Other Particulate Carriers In vivo Fate: Biodegradability andBiocompatibility All components are Some carriers have non-biomaterials, hence provide biological components that these propertiesimpair these properties In Vivo Fate: Toxicity and Immunogenicity Basedon nature of raw Varies from one carrier to materials, no toxicity andlow another. Acceptable in the to no immunogenicity are few systemsapproved for expected. In vitro and in clinical use. vivo studiesconfirm no toxicity. In Vivo Fate upon i.v. Administration: Retention inCirculation Good and sufficient retention Poor and insufficient wasconfirmed as the GAG retention obtained, unless component already hasthe carrier is surface-modified to hydrophilic outer shell found carryan appropriate ligand on to delay opsonization and its surface to delayboth uptake by the RES. opsonization and uptake by the RES.

The gagomers of the present invention also provide superior biologicaland therapeutic activity as compared with other particulate carriers.Some of these advantages are shown in Table 3.

TABLE 3 Advantages of Present Invention: Aspects ofBiological/Therapeutic Activity Gagomers Other Particulate CarriersBiological/Therapeutic Activity: Efficiency of Drug EntrapmentHigh-efficiency entrapment Entrapment efficiencies run independent ofdrug size up to from poor to high, with low and including proteins andefficiency of high molecular genetic material, due to a weight entities“wraparound” or “induced-fit” nature Biological/Therapeutic Activity:Site-Retention and Targeting The bioadhesive nature of the Furthercarrier modification, GAG component endows the which is not alwayspossible systems with ability to adhere and in some cases is counter-with high affinity to in vivo productive to production and recognitionsites and confers in vivo fate aspects, is measures of active targetingrequired to endow the systems with these properties.

Two basic type of gagomer may be synthesized: low lipid toglycosaminoglycan ratio, [1:1, w/w] denoted LLG, and high ratio of lipidto glycosaminoglycan, [5:1 to 20:1, w/w] denoted HLG. By changingspecific steps in the preparation, the outcome can be directed to formmicro- or nanoparticles.

The gagomers of the present invention, lipidated glycosaminoglycans, canbe used as delivery systems for drug therapy to treat a pathologicalcondition in an animal in need thereof. The term “animal” used herein istaken to include humans, and other mammals such as cattle, dogs, cats,rats, mice; as well as birds; reptiles; and fish.

For the present invention, pathological conditions suitable fortreatment by means of the gagomers include but are not limited tocancer, fungal or bacterial infections, including those secondary totrauma such as burns, infections caused by parasites or viruses, prioninfections, and the like.

The gagomers of the present invention may also have use in vaccinepreparations and gene therapy. The preparation of vaccines containing animmunogenic polypeptide as the active ingredient is known to one ofskill in the art. Likewise, the preparation of vectors for geneinsertion is also known to one of skill in the art.

The gagomers formed by the procedures of the present invention may belyophilized or dehydrated at various stages of formation. For example,the lipid film may be lyophilized after removing the solvent and priorto adding the drug. Alternatively, the lipid-drug film may belyophilized prior to hydrating the gagomers. Such dehydration may becarried out by exposure of the lipid or gagomer to reduced pressure,thereby removing all suspending solvent.

Alternatively or additionally, the hydrated gagomer preparation may alsobe dehydrated by placing it in surrounding medium in liquid nitrogen andfreezing it prior to the dehydration step. Dehydration with priorfreezing may be performed in the presence of one or more protectiveagents, such as sugars. Such techniques enhance the long-term storageand stability of the preparations.

Following rehydration, the preparation may be heated. Other suitablemethods may be used in the dehydration of the gagomer preparations. Thegagomers may also be dehydrated without prior freezing. Once thegagomers have been dehydrated, they can be stored for extended periodsof time until they are to be used. The appropriate temperature forstorage will depend on the lipid formulation of the gagomers andtemperature sensitivity of encapsulated materials.

When the dehydrated gagomers are to be used, rehydration is accomplishedby simply adding an aqueous solution, such as distilled water or anappropriate buffer, to the gagomers and allowing them to rehydrate. Thisrehydration can be performed at room temperature or at othertemperatures appropriate to the composition of the gagomers and theirinternal contents.

The gagomers of the present invention, lipidated glycosaminoglycans, arepreferably prepared by covalently binding a lipid having at least oneprimary amino group to a carboxylic acid-containing glycosaminoglycan bythe following method:

(a) A reaction vessel is provided in which the lipid is spread in a thinlayer on the vessel bottom and walls. This can be effected by dissolvingthe lipid in an organic solvent and evaporating the lipid to drynessunder low pressure in a rotary evaporator.

(b) The glycosaminoglycan is activated by pre-incubation in acidic pHwith a crosslinker.

(c) The activated glycosaminoglycan is added to the reaction vessel.

(d) The reaction mixture of the lipid and activated glycosaminoglycan isbuffered to a basic pH 8.6.

(e) The buffered reaction mixtures are incubated, with continuousshaking, for a period of time sufficient for the lipidatedglycosaminoglycan to form, such as overnight at 37° C. Since thelipidated gags are designed to be used in vivo, they should be stable atabout 37° C. While higher temperatures can be used for lipidation,lipids undergo physical changes with rising temperatures, generallyabout 62° C. Therefore, the lipidation preferably is conducted attemperatures from about 30-40° C.

(f) The lipidated glycosaminoglycan is buffered to a neutral pH andother ions and water-soluble additives are added according to need inorder to elevate the ionic strength to physiological levels with ions orsalts present in biological fluids (such as NaCl, KCl, Ca²⁺ and Mg²⁺).

(g) The particles are fractionated by successive centrifugations, eachrun at 4° C., for 40 minutes at the g force of 1.3×10⁵, as follows: Thepellet after 3 runs is the microparticle-enriched fraction, thesupernatant of the microparticle enriched fraction subjected to 3additional runs is the nanoparticle-enriched fraction.

(h) The resulting lipidated glycosaminoglycan is lyophilized.

To entrap drugs or other active ingredients in the gagomers, thematerial of interest is dissolved in ion-free pure water. Thelyophilized dry powder gagomer obtained as above is then reconstitutedin aqueous solution of the material to be entrapped.

Turbidity studies, following light scattering in a spectrophotometer,may be conducted for equal concentrations of soluble hyaluronic acid andof a gagomer prepared from hyaluronic acid and phosphatidylethanolamineto gain insight into whether the synthesis actually yields particulatematter. Representative results of such studies are shown in FIG. 5. Asexpected, over the concentration range tested free hyaluronic acid issoluble, and its solutions do not scatter light. In contrast, thegagomer-containing samples are turbid, the light scattering increasingwith the gagomer concentration, making it clear that the biopolymer isan insoluble material.

Samples of the gagomers entrapping macromolecules are viewable both bylight and by fluorescence microscopy. A typical field seen under thefluorescent microscope, of microparticles between 2 and 5 microns indiameter, prepared from HLG and entrapping a model protein, BSA-FITC, isshown in FIG. 6, top panel. These microparticles are prepared asdescribed above. Prior to viewing under the microscope, the nonentrappedprotein is removed from the preparation by ultracentrifugation at 4° C.for 30 minutes and a g force of 1.2×10⁵. The pellet containing theparticles with their entrapped protein is resuspended in phosphatebuffered saline (PBS).

A typical field of nanoparticles (between 50 and 200 nm in diameter)seen under the light microscope prepared from HLG entrapping a plasmidDNA is shown in FIG. 6, bottom panel. The nanoparticles are prepared asdescribed above for the FITC-BSA.

Particles made of glycosaminoglycans have a wide range of applications,as the same particles can be used alone, or with any type of materialencapsulated therein. The glycosaminoglycan particles are preferablymade without any encapsulated materials and then lyophilized to form apowder. The powdered glycosaminoglycan particles are then mixed with apowder of the material to be encapsulated. Alternatively, the powderedglycosaminoglycan particles are reconstituted by mixing with an aqueoussolution of the material to be encapsulated. Once the mixture isreconstituted, the particles will have captured the material that wasmixed in. Thus, small molecules, such as antibiotics andchemotherapeutic drugs, and large molecules, such as proteins, can beencapsulated with this technique. The particles can be used toencapsulate DNA, and the larger particles may even encapsulate wholecells and cell lines. Thus, the particles can also be used as a scaffoldfor tissue engineering.

The particles of the present invention are prepared by reacting at leastone glycosaminoglycan in the long form, i.e., the gag has not beensliced up into smaller sizes. All glycosaminoglycans, except hyaluronicacid, are naturally in the form of a protein moiety bound covalently toa poly-saccharide moiety. Methods for hydrolyzing the protein-sugar bondare well known to those skilled in the art, both chemically andenzymatically. In addition, some commercial products are available inwhich the protein moiety has already been removed.

The glycosaminoglycan polymer is reacted with a lipid which has at leastone primary amino group to cross-link the carboxylic residue of theglycosaminoglycan to a primary amine in the lipid. Once this reactionoccurs, thermodynamic stability causes the lipids to interact with oneanother so as to pull the product into a sphere having theglycosaminoglycan on the outside and the lipids on the inside. Theseparticles are then used to encapsulate other materials, including drugs,DNA, cells, proteins, etc.

In one embodiment of the present invention, the protein part of theglycosaminoglycan is removed and only the sugar backbone is reacted withthe lipids.

It is known in the art to attach hyaluronic acid to the outside ofliposomes for targeting or for making the liposomes more bioadhesive. Inthe instant invention, there is no liposome, rather, lipid molecules areattached covalently to hyaluronic acid.

In another embodiment of the present invention, other molecules may beattached first to the glycosamino-glycan, which is then reacted withlipids. These particles have the other molecules appearing on theoutside of the particles. These other molecules may be, for example,antibodies, folate, porphyrins, or lectins, and may be used fortargeting.

Although naturally-occurring glycosaminoglycans are preferred in thepresent invention in order to avoid problems with immunogenicity andtoxicity, synthetic glycosaminoglycans can be used, as well as natural,synthetic, or semisynthetic molecules, including but not limited tochondroitin, hyaluronic acid, glucuronic acid, iduronic acid, keratansulfate, heparan sulfate, dermatin sulfate, and fragments, salts, andmixtures thereof. The term glycosaminoglycan as used herein furtherencompasses glycosaminoglycans that have been chemically altered (butnot partially hydrolyzed), yet retain their function. Thesemodifications include, but are not limited to, esterification,sulfation, polysulfation, and methylation.

Natural sources of glycosaminoglycans include both plant and animalsources, including but not limited to beechwood trees and forms ofanimal cartilage, including shark cartilage, bovine trachea, whaleseptum, porcine nostrils, and mollusks such as Perna canaliculus and seacucumber.

It has been found that drugs encapsulated in the glycosaminoglycanparticles of the present invention are much more effective than the freedrugs, particularly for cancer cells that have become drug resistant. Itappears that the gagomers attach to the cancer cells and thus becomedepots of drugs which can enter the cells more quickly than they areexcreted. These drugs thus have a toxic effect on cells despite thedrug-resistant mechanisms that have been developed, overwhelming thecancer cells.

The gagomers of the present invention can encapsulate almost any type ofmolecule without being modified. In contrast, liposomes, for example,must first be positively charged in order to complex with DNA, whereasliposomes encapsulating many other materials are not positively charged.It is an advantage of the present invention that the gagomers canencapsulate virtually any type of molecule.

The glycosaminoglycans are used at sizes obtained when they are purifiedfrom their biological sources, and that have not been subjected tochemical and/or biological degradation. For example, for hyaluronicacid, this corresponds to a range of about 1×10⁵ to about 1×10⁷ daltons.

Pharmaceutical compositions using gagomers according to the presentinvention can be administered by any convenient route, includingparenteral, e.g., subcutaneous, intravenous, topical, intramuscular,intraperitoneal, transdermal, rectal, vaginal, intranasal orintraocular. Alternatively or concomitantly, administration may be bythe oral route.

Parenteral administration can be by bolus injection or by gradualperfusion over time. Parenteral administration is generallycharacterized by injection, most typically subcutaneous, intramuscularor intravenous.

Topical formulations composed of the gagomer constructs hereof,penetration enhancers, and other biologically active drugs ormedicaments may be applied in many ways. The solution can be applieddropwise, from a suitable delivery device, to the appropriate area ofskin or diseased skin or mucous membranes and rubbed in by hand orsimply allowed to air dry. A suitable gelling agent can be added to thesolution and the preparation can be applied to the appropriate area andrubbed in. For administration to wounds or burns, the gagomers may beincorporated into dosage forms such as oils, emulsions, and the like.Such preparations may be applied directly to the affected area in theform of lotions, creams, pastes, ointments, and the like.

Alternatively, the topical solution formulation can be placed into aspray device and be delivered as a spray. This type of drug deliverydevice is particularly well suited for application to large areas ofskin affected by dermal pathologies, to highly sensitive skin or to thenasal or oral cavities. Optionally, the gagomers may be administered inthe form of an ointment or transdermal patch.

Oral routes of administration are understood to include buccal andsublingual routes of administration.

The gagomers of the present invention may also be administered by otherroutes which optimize uptake by mucosa. For example, vaginal (especiallyin the case of treating vaginal pathologies), rectal and intranasal arepreferred routes of administration. Further, the gagomers areparticularly suited for delivery through mucosal tissue or epithelia. Ifadministered intranasally, the gagomers will typically be administeredin an aerosol form, or in the form of drops. This may be especiallyuseful for treating lung pathologies. Suitable formulations can be foundin Remington's Pharmaceutical Sciences, 16th and 18th Eds., MackPublishing, Easton, Pa. (1980 and 1990), and Introduction toPharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia(1985), each of which is incorporated herein by reference.

Depending on the intended mode of administration, the compositions usedmay be in the form of solid, semi-solid or liquid dosage forms, such, asfor example, tablets, suppositories, pills, capsules, powders, liquids,suspensions, or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. The pharmaceuticalcompositions will include the gagomer construct as described and apharmaceutical acceptable excipient, and, optionally, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, etc. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active compounds and which has no detrimentalside effects or toxicity under the conditions of use. The choice ofcarrier is determined partly by the particular active ingredient, aswell as by the particular method used to administer the composition.Accordingly, there are a wide variety of suitable formulations of thepharmaceutical compositions of the present invention.

Suitable excipients are, in particular, fillers such as saccharides, forexample, lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch paste using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidine.

Injectable formulations for parenteral administration can be prepared asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate, etc.

Aqueous injection suspensions may also contain substances that increasethe viscosity of the suspension, including, for example, sodiumcarboxymethylcellulose, sorbitol, and/or dextran. Optionally, thesuspension may also contain stabilizers.

The parenteral formulations can be present in unit dose or multiple dosesealed containers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, e.g., water, for injections immediately prior touse. Extemporaneous injection solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed.

For oral administration, a pharmaceutically acceptable, non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example, mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, sodiumcrosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and thelike. Such compositions include solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations and the like. Formulations suitable for oral administrationcan consists of liquid solutions such as effective amounts of thecompound(s) dissolved in diluents such as water, saline, or orangejuice; sachets, lozenges, and troches, each containing a predeterminedamount of the active ingredient as solids or granules; powders,suspensions in an appropriate liquid; and suitable emulsions. Liquidformulations may include diluents such as water and alcohols, e.g.,ethanol, benzyl alcohol, and the polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptable surfactant,suspending agents, or emulsifying agents.

When the composition is a pill or tablet, it will contain, along withthe active ingredient, a diluent such as lactose, sucrose, dicalciumphosphate, or the like; a lubricant such as magnesium stearate or thelike; and a binder such as starch, gum acacia, gelatin,polyvinylpyrolidine, cellulose and derivatives thereof, and the like.

Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, crosscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other preservatives, flavoring agents, and pharmaceuticallyacceptable disintegrating agents, moistening agents preservativesflavoring agents, and pharmacologically compatible carriers.

Capsule forms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricant, and inert fillers, suchas lactose, sucrose, calcium phosphate, and corn starch.

Lozenge forms can comprise the active ingredient in a carrier, usuallysucrose and acacia or tragacanth, as well as pastilles comprising theactive ingredient in an inert base such as gelatin or glycerin, orsucrose and acacia.

In determining the dosages of the gagomer particles to be administered,the dosage and frequency of administration is selected in relation tothe pharmacological properties of the specific active ingredients.Normally, at least three dosage levels should be used. In toxicitystudies in general, the highest dose should reach a toxic level but besublethal for most animals in the group. If possible, the lowest doseshould induce a biologically demonstrable effect. These studies shouldbe performed in parallel for each compound selected.

Additionally, the ED50 (effective does for 50% of the test population)level of the active ingredient in question should be one of the dosagelevels selected, and the other two selected to reach a toxic level. Thelowest dose is that dose which does not exhibit a biologicallydemonstrable effect. The toxicology tests should be repeated usingappropriate new doses calculated on the basis of the results obtained.

Young, healthy mice or rats belonging to a well-defined strain are thefirst choice of species, and the first studies generally use thepreferred route of administration. Control groups given a placebo or nottreated are included in the tests. Tests for general toxicity, asoutlined above, should normally be repeated in another non-rodentspecies, e.g., a rabbit or dog. Studies may also be repeated usingalternate routes of administration.

Single dose toxicity tests should be conducted in such a way that signsof acute toxicity are revealed and the mode of death determined. Thedosage to be administered is calculated on the basis of the resultsobtained in the above-mentioned toxicity tests. It may be desired not tocontinue studying all of the initially selected compounds.

Data on single dose toxicity, e.g., LD50, the dosage at which 50% of theexperimental animals die, is to be expressed in units of weight orvolume per kg of body weight and should generally be furnished for atleast two species with different modes of administration. In addition tothe LD50 value in rodents, it is desirable to determine the highesttolerated dose and/or lowest lethal dose for other species, i.e., dogand rabbit.

When a suitable and presumably safe dosage level has been established asoutlined above, studies on the drug's chronic toxicity, its effect onreproduction, and potential mutagenicity may also be required in orderto ensure that the calculated appropriate dosage range will be safe,also with regard to these hazards.

Pharmacological animal studies on pharmacokinetics revealing, e.g.,absorption, distribution, biotransformation, and excretion of the activeingredient and metabolites are then performed. Using the resultsobtained, studies on human pharmacology are then designed.

Studies of the pharmacodynamics and pharmacokinetics of the compounds inhumans should be performed in healthy subjects using the routes ofadministration intended for clinical use, and can be repeated inpatients. The dose-response relationship when different doses are given,or when several types of conjugates or combinations of conjugates andfree compounds are given, should be studied in order to elucidate thedose-response relationship (dose vs. plasma concentration vs. effect),the therapeutic range, and the optimum dose interval. Also, studies ontime-effect relationship, e.g., studies into the time-course of theeffect and studies on different organs in order to elucidate the desiredand undesired pharmacological effects of the drug, in particular onother vital organ systems, should be performed.

The compounds of the present invention are then ready for clinicaltrials to compare the efficacy of the compounds to existing therapy. Adose-response relationship to therapeutic effect and for side effectscan be more finely established at this point.

The amount of compounds of the present invention to be administered toany given patient must be determined empirically, and will differdepending upon the condition of the patients. Relatively small amountsof the active ingredient can be administered at first, with steadilyincreasing dosages if no adverse effects are noted. Of course, themaximum safe toxicity dosage as determined in routine animal toxicitytests should never be exceeded.

Compositions within the scope of the present invention include allcompositions wherein the active ingredient is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each compound iswithin the skill of the art. The dosage administered will depend uponthe age, health, and weight of the individual recipient thereof as wellas upon the nature of any concurrent treatment and the effect desired.Typical dosages comprise 0.01 to 100 mg/kg body weight. The preferreddosages comprising 0.1 to 100 mg/kg body weight. The most preferreddosages comprise 1 to 50 mg/kg body weight.

The gagomers may be formulated to entrap therapeutic compositions fordrug or gene therapy, or may be empty, for use in treating cancer,especially metastatic cancer.

EXAMPLE 1 Structural Studies of Micro-Gagomers

The structural data provided here (FIGS. 1A and 1B) was obtained bymeans of Scanning Electron Microscopy (SEM). Both parts of FIG. 1 arefields from the same batch, at two different magnifications (seeinformation stamped by the device itself at the bottom of each figure).Three features are demonstrated by these results: (1) these dataconstitute a confirmation of the particulate nature of these polymers;(2) these data also constitute a confirmation of the size range (see 1μm bar in FIG. 1A); and (3) some details are provided on the shape ofthe particles.

The particles are seen to be heterogeneous with respect to size. This isseen in FIG. 1A and more so in FIG. 1B. This is an expected outcome,since microscopy was done on the whole preparation, prior tofractionation into the nano- and microparticle populations. Themagnification ranges applied under the microscope for use in SEM favorthat of the microparticles.

EXAMPLE 2 Chemical Bonding

Since the lipid amino group was crosslinked to the carboxylic residuesof hyaluronic acid, there should be a decline in the number of freecarboxylic acids from free hyaluronic acid to the gagomer. The morelipid bound, the more extensive should be the decline of free carboxylicacid groups. Moreover, from the extent of free carboxylic acid loss, itis possible to measure the lipid to hyaluronic acid stoichiometry. Usinga carboxylic acid assay, it was possible to measure the expecteddecline. It could also be estimated that, in the microparticles, about33% of the glucuronic acid residues are occupied by lipid molecules,wherein in the nanoparticles only about 20% of the glucuronic acidresidues are occupied by lipid molecules.

Example 3 Physicochemical Details and Properties of the EtBr GagomerFormulation

The efficiency of entrapment of drugs or other bioactive agents in thegagomers and the kinetics of drug efflux for small molecular weightdrugs were determined using absorbency in an ELISA plate reader, withappropriate wavelengths for each given entrapped entity.

Typical results of the efficiency of entrapment are shown in Tables 4and 5 of the microparticles and nanoparticles, respectively.

TABLE 4 Micro-Gagomers: Efficiency of Drug Entrapment and Half-Life ofDrug Efflux Encapsulation Efficiency (%) Half-Life of Entrapped By DrugEfflux Entity Thermodynamics By Kinetics (Hours) Fluorescein 49.6 ± 4.840.6 ± 5.8 7.9 Chloramphenicol 39.3 ± 3.9 30.5 ± 1.9 28.2 Mitomycin C49.4 ± 2.5 44.3 ± 2.7 20.1 Doxorubicin 52.4 ± 6.3 50.2 ± 1.2 35.3 BSA32.0 ± 2.5 DNA 74.5 ± 2.8

TABLE 5 Nano-Gagomers: Efficiency of Drug Entrapment and Half-Life ofDrug Efflux Encapsulation Efficiency (%) Half-Life of Entrapped By DrugEfflux Entity Thermodynamics By Kinetics (Hours) Fluorescein 37.4 ± 1.229.1 ± 6.1 21.9 Chloramphenicol 47.4 ± 0.3 47.1 ± 1.5 14.8 Mitomycin C54.8 ± 0.9 41.7 ± 1.6 29.8 Doxorubicin 57.0 ± 3.7 53.6 ± 0.9 22.3 BSA35.0 ± 1.8 DNA 65.8 ± 4.8

The concentration of gagomer-entrapped EtBr was 25 μM. Efficiency ofentrapment was 49.8(±3.1) (%). Half-life of EtBr efflux from the gagomerwas 27.7 hours.

Example 4 In Vitro Toxicity Studies

Drug-free gagomers of both micro- and nano-size ranges, were tested fortoxicity in cell cultures for both low lipid and high lipid gagomers.Two cell lines were tested, the rat glioma cell line C6 and the mousefibroblast line NIH3T3. In all cases the gagomers were found to have notoxicity over the 100-fold concentration range of 0.02 to 2 mg/mlpolymer.

EXAMPLE 5 Therapeutic Activity Exemplified by Treatment of aDrug-Resistant (MDR) Glioma Cell Line

Due to their location and poor response to chemotherapeutic drugs, braintumors, particularly gliomas, are very difficult to treat (Wolff et al,1999; Nutt et al, 2000. The poor drug response is due in part to lack ofaccess and in part to inherent multidrug resistance (MDR) of thesetumors (Larsen, 2000; Gottesman et al, 1995).

In brain tumors, multidrug resistance is an impediment even in caseswhere access to the tumor has been provided, such as by localadministration or leaving a local depot at the end of a surgicalprocedure. In this prevalent drug resistance mechanism, which appears inboth an acquired and inherent mode, the drugs do not lose theirintrinsic toxic activity, nor have the resistant cells found a way tometabolize the drugs to nontoxic entities. Rather, the drug that entersthe cell through passive diffusion across the cell membrane is activelypumped out, reducing intracellular levels to below their lethalthreshold. The glioma C6 line, which displays inherent MDR, served asthe model system for testing whether treatment with gagomersencapsulating a chemotherapeutic drug would offer any advantage over asimilar treatment with the free drug.

Methodology

Cells were seeded onto 96 well plates, and the experiment was initiatedat semi-confluency, usually 24 hours post seeding. The cells were givena selected dose of the drug of choice, entrapped in a gagomerformulation that was washed of excess nonentrapped drug prior to use.Control systems were the same dose of free drug, and a dose of drug-freegagomer at a dose similar to that of the test system. Cell survival wasdetermined 48 hours post-treatment, using the MTT assay (Nutt et al,2000; Larsen et al, 2000).

Results

Results for three chemotherapeutic drugs are shown in FIG. 8 in threedata sets. The data for the free gagomer (left-most bar in each of thethree data sets) is an additional confirmation of the data discussedabove with respect to the gagomers being non-toxic. Depending upon thespecific drug, with each drug operating at its own dose range, it can beseen that even relatively high doses of free drug permit 20-60% of thecells to survive. Such results, shown in the middle bar of each dataset, are typical for the inherent form of multidrug resistant cells.Replacing the free drug with the same dose of gagomer-entrapped druggenerated a dramatic difference, as can be seen by the right-most bar ineach data set. For each of the three drugs, the novel formulationgenerates a 3-4-fold increase in cell death as compared to thecorresponding free drug. Two findings tightly link this improvedresponse in treatment to the novel drug delivery formulation of thepresent invention: the non-toxic nature of the free gagomer, and theincreased cell demise obtained for three different drugs that each havea unique cytotoxic mechanism.

To overcome multidrug resistance, a mechanism must be found to elevateintracellular doses of a chemotherapeutic drug above the lethalthreshold. The traditional approach taken in the attempt to achieve thiselevation is to reduce the pumping by using reversal agents that arealso known as chemosensitizers. While several of these agents have beenidentified, most prominent among them verapamil, none of the currentlyavailable chemosensitizers can be used clinically. In addition,treatment requires careful orchestration, as the two active entities,the chemotherapeutic drug and the chemosensitizers, must reach thetarget together to be effective. This is not a simple matter in clinicalpractice.

Another way to elevate intracellular drug dose is to increase influx,both in magnitude and duration. It appears that the outstanding increasein drug response for the drug-entrapping gagomers of the presentinvention operates by increasing influx. The bioadhesive nature of thegagomers positioned them as drug depots bound to the cell membrane. Thisboth increased the electrochemical gradient of the drug across the cellmembrane as compared to the free drug, as well as the time span duringwhich drug entry occurs. Thus, treatment only requires one entity, thedrug-gagomer composition. These new formulations will also benefitnon-resistant tumors by allowing successful treatment with significantlylower drug doses.

EXAMPLE 6 Interaction of Micro-Gagomers with Cells

Cells are known to be impermeable to EtBr (ethidium bromide), anucleic-acid sensitive fluorescent marker. Its fluorescence emission issignificantly enhanced upon binding to DNA and RNA, allowing fordetermination of whether a carrier has made cells permeable to EtBr and,in particular, whether it has reached the nucleus.

In order to probe the interactions of these novel polymers with cellsEtBr-encapsulating gagomers were prepared, the physicochemicalproperties of these gagomers were determined, and then the gagomers wereincubated with cells. The results were scanned using confocalmicroscopy.

Two cell lines were tested, C6—a rat glioblastoma cell line, andPANC-1—a human pancreatic adenocarcimona cell line. For each cell line,monolayers of the cells were incubated with three differentformulations: (1) free EtBr, (2) “empty” (i.e. encapsulating bufferalone) gagomers suspended in a solution of free EtBr, and (3)EtBr-entrapping gagomers.

In all three formulations the EtBr was used at the same 25 μMconcentration. The gagomers in formulations (2) and (3) were at the sameconcentration—0.25 mg/ml. Each formulation was incubated with the cellsfor 60 minutes at room temperature, prior to performance of the confocalmicroscopy. The results are shown in FIG. 2 for the cell line C6 and inFIGS. 3 and 4 for the cell line PANC-1.

The results for the C6 cell line is shown in FIG. 2A. The upper leftsection shows results of cells incubated with free EtBr. It is clearthat there is negligible fluorescence inside the cells, as expected forthis marker when it is free in solution. The upper right section of FIG.2A is for the cells incubated with free EtBr in a solution that hadempty gagomers suspended in it. Negligible fluorescence is seen here,and its similarity to free EtBr is a clear indication that the particlesthemselves do not promote entry of free (non-entrapped) EtBr into thecells.

In contrast to these two controls, when the EtBr is entrapped inside theparticle, it gains entry into the cells and into the nucleolus. This isclear from the high fluorescence intensity of the bottom part of FIG.2A, and from its localization inside the cells inside the nucleolus(interacting with DNA) and also in the cytosol (interacting with RNA).These findings are not restricted to a specific cell line, as similarresults were obtained with the PANC-1 line also.

In FIG. 3 the results with formulations (2) and (3) alone are depicted.The sizeable differences between free and gagomer-entrapped EtBr areshown in greater detail in FIG. 4. FIG. 4A shows a single cell incubatedwith free EtBr. Only a negligible amount of the marker has entered thecell and reached the nucleolus. Also, if there is any EtBr in thecytosol it is below detection. In contrast, as shown in FIG. 4B, whenincubated with gagomer-entrapped EtBr, substantial amounts of the markerenter the cell and are found in the nucleus (DNA-bound) and in thecytosol (RNA-bound).

As all data were obtained using the same concentration of EtBr, it seemsclear that the entrapment within the polymer made the difference. Inprinciple, there are three major mechanisms that can account for acarrier facilitating entry of its nucleic acid-sensitive marker loadinto a cell in such a manner that allows free intracellular marker tointeract with RNA and also gain entry into the nucleolus to interactwith the DNA:

(1) Adsorption and Diffusion. The marker-loaded particles adhere to thecell membrane, creating local depots. Marker diffuses out of theadhering particles and some of this freed marker diffuses across thecell membrane, into the cell.

(2) Fusion. The marker-loaded carrier first binds to the cell membrane,then fuses with it and in the course of fusion, entrapped material isreleased into the cytosol.

(3) Endocytosis and Release. The marker-loaded carrier enters the cellby an endocytotic pathway. The endocytosed carrier succeeds in releasingmarker into the cytosol. In all three mechanisms, once the marker isfree in the cytosol, part of this now-intracellular marker pool findsits way to the nucleolus.

The first mechanism may be eliminated on account of physicochemical datashowing efflux of the entrapped marker to be quite slow. Based on theefflux rate constant (listed above in the form of half-life), it can becalculated that in the course of the 50 minutes incubation prior to themicroscopy, efflux would be at the most 2% of the entrapped marker,corresponding to 0.5 μM EtBr becoming free. Even if all of this were toget across the cell membrane into the cell, the result would have beeneven more negligible than seen with the 50 fold higher concentration (25μM vs. 0.5 μM) of free EtBr (FIG. 2A). In contrast, the results with thecarrier-entrapped marker show a substantially higher entry such as couldnot be obtained through the “adsorption and diffusion” mechanism.

Regardless of whether the fusion or endocytotic mechanism of treatmentof cancer cells is the means by which entrapped marker enters the cell,it is clear that this carrier allows impermeable molecules into the celland into the nucleolus. This ability bodes well for performance of thegagomers in drug delivery.

EXAMPLE 7 Formulation Studies Particle Properties

Sizing the Particles: The low lipid to glycosaminoglycan ratio (LLG) andhigh lipid to glycosaminoglycan ratio (HLG) nano- and microparticleswere sized using an ALV-NIBS particle sizer. The results, listed inTable 6, provide full quantitative data and are in good agreement withthe previously-obtained microscopy data (EM, fluorescence). The twosizes are well distinguished from one another, and the relatively lowscatter within each system indicates good efficiency of the separationprocess. The data also show that within each particle type, there issome flexibility in designing particle size through manipulation of thelipid/HA ratio.

TABLE 6 Size Distributions of the Nano and Micro Tau DDS SystemsParticle Specifications Particle Diameter Type Lipd/HA Ratios (nm) NanoLLG 227 ± 37 HLG 135 ± 41 Micro LLG 1330 ± 225 HLG 1150 ± 178

Zeta Potentials: The zeta potentials of both micro- and nanoparticleswere measured, as a function of particle concentration. The zeta, orelectrokinetic potential represents the potential across the diffuselayer of ions surrounding any charged colloidal particle, and is largelyresponsible for colloidal stability. Typical results, shown in FIG. 9,demonstrate that: (a) as expected on the basis of particle chemicalcomposition and particle structural features, the zeta potentials arenegative; and (b) the patterns observed for the dependence of zetapotential on concentration fit with the general pattern observed in thefield for negatively charged particles.

Entrapment Efficiencies

Two formulations were investigated: insulin and α-interferon, eachentrapped in separate formulations in the microparticles. The entrapmentefficiencies obtained are shown in Table 7. Clearly, both new proteinsare entrapped with high efficiency, as was previously shown for othermacromolecules (Tables 4 and 5). The insulin concentration was 10 mg/ml.At this range this protein is already aggregated into dimers andhexamers, meaning that the entities entrapped were larger than 6000 da.Levels of encapsulation this high, at this level of insulin doses, werenot reported for other particulate carriers.

TABLE 7 Encapsulation Efficiencies of Therapeutic Proteins in the NovelDDS (Microparticles) MW Range Encapsulation Efficiency EncapsulatedMatter (Da) (%) Insulin 6,000 86.9 ± 4.7 (Human Recombinant)α-Interferon 19,000 72.5 ± 3.7 (Human Recombinant)

EXAMPLE 8 In Vitro Studies Toxicity Testing in Cell Cultures

Toxicity testing of the free DDS was done as follows: cells of a givenline were incubated with increasing concentrations of the DDS, spanningthe range of 0.01-5 mg/ml, for 24 or 48 hours. Control cells were notexposed to the DDS. These tests were performed with 8 different celllines, originating from human, rat and mice. The common feature of alleight cell lines was that they have receptors for hyaluronic acid.Results similar to those for the DDS dose of 1 mg/ml, as shown in FIG.10, were over the whole DDS concentration range tested (i.e., 0.01-5mg/ml). The data in FIG. 10 demonstrate that over all the DDS doses,incubation periods and cell lines this DDS is not toxic to cells.

Gene Transfection

The ability of the DDS to entrap plasmids at exceptionally highaffinity, was reported in Tables 4 and 5. The potential of such aformulation to transfect cells with a desired plasmid that would resultin expression of the encoded protein was tested in vitro.

The cell lines tested were PANC-1 and C6, both cell lines with receptorsfor hyaluronic acid. The reporter gene was the one encoding for GreenFluorescent Protein (GFP). The DDS was compared to twocommercially-available vectors that served as “benchmarks”: Polyplex—acationic polymer, and lipofectamine—a cationic liposome. Protocols usedfor the commercial vectors were those recommended by the manufacturers.

The plasmid was entrapped in the DDS (microparticles), and was allowedto equilibrate for 24 hours prior to use. The DNA concentration was thesame for all three vectors, 1.5 pg/well. Cells were incubated with theselected vector-DNA formulations in DMEM for 5 hours; control wells wereincubated for the same period in DMEM alone. At the end of 5 hours,serum-supplemented cell growth media was added to all wells.

Cells were viewed under an inverted fluorescent microscope at 12 and at24 hours from the starting point. The total number of cells, and thenumber of fluorescent cells in the viewed sample were counted. Thesedata were used to calculate the transfection efficiency, defined as the% fluorescent cells of the total cells, in the viewed sample. The totalnumber of cells in a viewed sample was 200-400 cells. Cell viability wastested upon termination of the experiment, 24 hours from the startingpoint.

The results obtained are listed in Table 8. The doses used for thebenchmarks were 2 mg/ml (as recommended in their established protocols)and for the DDS 0.2 mg/ml was used, a ten fold lower dose. The DNAconcentration was the same for all three. At 12 hours the gene product,GFP, was detected with the established vectors. This finding, althoughexpected, was encouraging since the cell lines tested were those ofspecial therapeutic interest, but not the classical cell lines (such asCOS 7) used in transfection. With the DDS, 24 hours was required forprotein expression. The transfection efficiencies for each of the threevectors, in both cell lines, also listed in the table, show that theperformance of the DDS vector works as well as the bench marks, at atenth of the dose (0.2 mg/ml vs. 2 mg/ml).

TABLE 8 In Vitro Gene Transfection Transfection Efficiency Time to at 24Hours Cell Viability (% Vector Detection (%) from Untreated Speciesmg/ml (hours) PANC-1 C6 Control) Polyfect 2 12 18 20 <50 Lipofectamine 212 12 15 <50 DDS 0.2 24 19 19 100

One of the severe drawbacks of gene transfection vectors that arecationic polymers or cationic lipids is toxicity. This was observed forthe two established vectors here—in each cell line, the level of viablecells at 24 hours was less than 50% compared to the untreated control.In contrast, there was no toxicity with the DDS vector. Cell viabilityremained as high as that of the control cells. The toxicity datareported in the previous section suggests that the DDS doses elevated tothose of the established vectors, 2 mg/ml, would also not have beentoxic.

The data clearly support the potential application of the novel DDS ingene therapy. There appear to be two distinct advantages over competingnon-viral vectors: (1) in two different cell lines it was as good asestablished vectors at a 10 fold lower concentration to achieve the samelevel of protein expression, suggesting that for equal vectorconcentrations the DDS system may be significantly superior to itscompetitors; and (2) in two different cell lines, no toxicity occurredwith the DDS vector system, whereas the other two vectors used werequite toxic.

Treatment of MDR Tumors

In cell culture studies designed to evaluate the cytotoxicity of adrug-entrapping targeted carrier, compared to the same dose of freedrug, the experimental design—and therefore the results—is usuallybiased in favor of the free drug. This is due to the static vs. dynamicconditions, for the in vitro and in vivo situations, respectively. Invitro, the free drug is in continuous contact with the cells for theduration of the experiment, usually 24 hours or more. In vivo durationof drug—administered in free form—at the tumor site will be muchshorter, due to the limited time span of administration and the naturalclearance processes. In vitro performance of a drug/carrier formulation(even a targeted carrier) may not be much different than that of thefree drug, under incubation periods of 24 hours or more. In contrast, invivo—if the carrier adheres to the target and stays there as a sustainedrelease depot, drug supply to the tumor site may be much higher (doseand duration) than the free drug, resulting in enhanced cytotoxicity.

In order to reduce the in vitro bias in favor of free drug, cells wereexposed to the following treatment formulations: free drug, drugentrapped in the DDS, and free DDS for a period of only 4 hours. Thetreatment media was then replaced with serum-supplemented cell growthmedia free of any drug or carrier, and the number of viable cells wasdetermined 20 hours later (24 hours from start). If some of the carrierformulation adhered to the cells, it should remain there as a depotdespite replacement of the media, and continuously feed the cells withdrug while the cells that received free drug would not be exposed to anymore drug, once the media was replaced.

Typical results showing the increase in cell death (compared tountreated control) as a function of treatment formulation, are shown inFIG. 11, for the cell line C26. This is an inherent multidrug resistant(MDR) line originating from mouse colon carcinoma. In FIG. 11A theresults with the drug mitomycin C (MMC) are shown. As expected from theprevious in vitro toxicity studies (FIG. 2), free DDS (tested here withthe dose of 1 mg/ml) was not toxic. Two doses of free MMC 30 and 50μg/ml were hardly effective, resulting in cell death percentages ofunder 15%. This low response to rather high doses is a manifestation ofthe MDR nature of these cells. In contrast, when treatment was with thesame drug doses but entrapped in the DDS, 80-100% of the cells werekilled. The differences in response for each drug dose—carrier-mediatedvs. free—are highly significant (p<0.001). Similar results are shown inFIG. 11B for another drug, doxorubicin (DOX). Free drug (each drugspecies has a different dose range) is ineffective while the same dosesformulated in the carrier were highly effective, also generating 80-100%cell kill. Comparable results were obtained with two other cell lines—C6and PANC-1. All three line tested have HA receptors.

EXAMPLE 9 In Vivo Studies

In Vivo Studies I: Tumor Chemotherapy

Female BALB/c mice which were 8 weeks old at initiation of theexperiment were used. The tumor model employed was C-26 cells(originating from mouse colon carcinoma) injected subcutaneously intothe right hind footpad. The chemotherapeutic drug was mitomycin C (MMC)free, or entrapped in DDS, LLG, in nanoparticulate form. The MMC dosewas 2 mg/kg body, in both free and DDS formulations and the DDS dose was1 mg/ml.

Experimental Design for Run 1

The experiment was performed with 20 animals, divided into 4 groups,each group of 5 mice receiving a specific treatment as listed in Table9, below.

TABLE 9 Animal Groups Group # Treatment 1 Saline 2 Free DDS 3 Free MMC 4MMC/DDS

To provide the tumor, C-26 cells were grown in cell culture flasks. Atday zero, the cells were harvested, washed several times, counted andimmediately injected. The injected dose was 8×10⁵ cells in 30 μl.

Treatments were given on days 5, 12 and 19. Administration was byinjection into the tail vein. All injected volumes were 0.1 ml.

Experimental Design for Run 2

Experimental design was essentially similar to that of Run 1, with thefollowing changes:

-   -   a. Drug dose was elevated to 5 mg/ml.    -   b. Tumor inoculation dose was 8×10⁵ cells in 30 μl.    -   c. The experiment was conducted with 2 groups, one receiving        free DDS and the other MMC/DDS, with 3 and 5 mice per group,        respectively.    -   d. Treatment were given on days 14, 17, 20 and 23.    -   e. Tumor size at initiation of treatment was 75 mm³.

Parameters measured for Run 1 were retention in circulation, tumoronset, tumor volume, survival. Parameters measured for Run 2 weresurvival.

Results for Run 1: Retention in Circulation

The reticuloendothelial system (RES) as part of its normal physiologicalprocesses, operates to remove foreign particulate matter from thecirculation rather swiftly. Unless the target of an intravenously (i.v.)administered particulate carrier is within the RES, this removal is amajor problem for all i.v.—administered particulate carriers, since theit reduces the likelihood of a sufficient dose reaching its intendedtarget in an efficacious manner. This problem is not specific to tumortreatment. It is general for any pathological situation that requiresi.v. administration.

Through extensive studies, means to block this process, thus allowingfor long-term circulation of particulate matter, sum up to the followingcombination: the particle should be small and it should have ahydrophilic coat, usually due to an abundance of hydroxyl residues.Particulate carriers of the sphere type—made on the nano scale(nanospheres)—are usually coated by polymers such as poloxomar orpoloxamine. Small liposomes usually carry polyethylene glycol (PEG) ontheir surface, and come under names such as “stealth liposomes”,“PEGylated liposomes” and “sterically-stabilized liposomes”.

Upon commencing the invention and development of the present DDS, it washypothesized that due to hyaluronic acid being its major component thesurface of the particle will be rich in hydroxyl residues that willprovide it with an intrinsic ability of long retention in circulationand with targeting ability. These would be distinct advantages over thecompetitive carriers, as both targeting and “stealth” properties arealready built in.

At selected periods post-injection, animals receiving drug-containingformulations were bled, and samples were treated according toestablished protocols. MMC concentration was determined by HPLC assay.Typical results of the retention in circulation, comparing free MMC toMMC entrapped in the carrier (MMC/DDS) are shown in FIG. 12. The datashow that free MMC disappears very quickly from the circulation, whereasMMC administered in the carrier circulates for a much longer period oflong time. This finding was reproduced from one injection to another,and drug was found in the circulation when administered via the carrierup to 72 hours post-injection. The fast disappearance of free drugindicates that the MMC found in the circulation of the animals receivingthe MMC/DDS formulation is in the carrier. These results confirm thehypothesis, discussed above, that these DDS have intrinsic “stealth”capability. As indicated above, this carries positive implicationsbeyond the specific pathology tested here.

Results for Run 1: Tumor Onset and Tumor Volume

Results of the increase in tumor volume, for all 4 groups, are shown inFIG. 13, together with the average day on which tumors were firstdetected. In all animals receiving saline alone, tumor was detected onday 7, and it increased fast and exponentially. Tumor was detected onday 7 in all animals receiving free drug as well. The increase in tumorvolume, despite receiving 3 doses of a chemotherapeutic drug, was notmuch different than in the saline group. This indicates that the MDRnature of this cell line previously seen in vitro (FIG. 3) also persistsin vivo. Surprisingly, treatment with free DDS was better than salineand than free drug. Average day of tumor appearance was 9 (vs. 7), andthe tumor growth rate was distinctly slower. Tumors were alsosignificantly smaller compared to the saline and free drug groups.

The performance of the free DDS in vivo is quite different from thatobserved in vitro. Hyaluronic acid is one of the key components ofextracellular matrix (ECM) and it is known that tumor cells that havereceptors for HA make use of this. Through interaction of their HAreceptors with the HA in the ECM, the tumor cells may use the ECM as aplatform in the course of tumor progression. Blocking the receptors may,therefore, delay tumor progression. This could be a major mechanismresponsible for the results obtained with free DDS, where the carrierbinds to HA receptors and is able to block them. Other potentialmechanisms, not mutually exclusive, are performance of the free DDS asan anti-angiogenic factor or as a general boost to host-defensemechanisms. The mechanisms responsible for this positive effect of theDDS itself will be pursued in order to understand these phenomena andlearn how to exploit them for better therapeutic outcomes. Regardless ofits origins, this is a positive additional advantage of this DDS, whichwas not anticipated on the basis of the in vitro data.

The best results were obtained with the drug entrapped in the carrier.As seen in FIG. 13, tumor was first detected on about day 17, much laterthan in the groups treated with free DDS, free drug, or saline. Tumorgrowth rate was slowest and tumors were smallest, of all groups tested.Perhaps this is due to the intrinsic targeting of this DDS wherein thefraction that reached the tumor remained there, acting as a drug depotand possibly combining the cytotoxicity effect of drug and the carriereffect seen with free DDS. The in vitro results that showed that thisformulation, unlike free drug, was capable of killing MDR cells, werethus repeated and confirmed in the in vivo case also.

Results for Run 1: Survival

Animal survival was monitored for over 90 days until the last animaldied. The results are shown in FIG. 14.

All animals from the groups receiving saline died between days 29 and31, and those receiving free drug died between days 31 and 33. Theanimals receiving the free DDS survived twice as long as the saline andfree drug groups, dying between days 59 and 66.

This long survival carries two critical implications. The first is thatthis DDS has no in vivo toxicity as was previously shown in vitro. Theweight of the in vivo evidence is much more significant in itsimplications for all applications of this technology. The secondimplication is that, concurrent with the effect on tumor development andsize (FIG. 13), the free carrier by itself has a beneficial therapeuticeffect on tumor bearing animals.

The longest survival, 3 times as long as for the saline and free druggroups, was observed for the animals receiving the full treatment, thedrug entrapped in the DDS. The last animal died on day 94. This isexceptionally long survival for tumor-bearing mice, especially in an MDRcase, and indicates the superiority of this drug delivery technologycompared to its competitors.

Results for Run 2: Survival

Animal survival was still being monitored on day 91 of the experiment,and the results are shown in FIG. 15. The three tumor-bearing animalstreated with the free DDS were the longest survivors, up to 69 days. Thefive tumor-bearing animals treated with the MMC/DDS formulation faredeven better, as at 91 days post tumor inoculation all animals werealive.

The trend of these data is similar to that obtained in Run 1, showingthat the exceptional responses to the novel DDS are reproducible. Twomajor differences existed between the two experiments. First, in Run 2the treatment was initiated after the tumor was developed (seeexperimental design above), which makes it a more challengingtherapeutic situation compared to Run 1. Secondly, in Run 2 the animalsreceived a higher cumulative drug dose. There were 4 injections (vs. 3in Run 1) and the dose was 2.5 fold higher (5 vs. 2 mg/ml).

The positive trend these differences induced indicates a potential togenerate even better responses with the novel DDS. More challenging butalso more realistic models, in which the tumor grows up to the sizerange of 100-150 mm³, before treatment is initiated may be amenable tothe novel DDS approach.

EXAMPLE 10 In Vivo Studies II: Intranasal Delivery to the Brain

Treatment of neurodegenerative diseases requires drug delivery to thebrain, either crossing an intact BBB or bypassing it. Two experiments,one in rats and the other in mice, were conducted to evaluate theability of the novel DDS of the present invention to deliver drugs tothe brain, bypassing the BBB via intranasal (IN) administration.

Run 1 comprised a rat experiment. Healthy pigmented rats were used. TheDDS was LLG, in nanoparticulate form and the marker was MMC. The testsystem was the marker formulated in the novel DDS. The dose administeredwas 5 mg/kg body, in both free and DDS formulations, 300 μl/animal. TheDDS dose was 1 mg/ml.

The experiment was conducted with 4 animals, divided into two pairs. Onepair received the free marker, intranasally (IN), into the rightnostril. The other pair received the marker/DDS formulation, IN, intothe right nostril. Administration was slow, over several minutes, usingan appropriate needle-less syringe.

At 6 hours post administration, the animals were sacrificed and thebrains were removed. Each brain was soaked in 10 ml PBS for an hour, todesorb loosely attached marker, after which the brains were homogenized.The marker was assayed in the wash and in the brain homogenates, usingan HPLC assay.

The results obtained are shown in FIG. 16, wherein the markeraccumulation is presented as % from administered dose. Even though therewere only 2 animals per treatment group, the agreement within each groupwas good enough to allow averaging. The average and standard deviationfor each pair, in the wash and in the brain homogenate, are listed abovethe relevant bars.

Focusing on the brain homogenate, marker accumulation in the brain whenadministered in free form was negligible, on the order usually seen withfree small molecules, as was expected. In contrast, when administered inthe DDS form, there was substantial accumulation of the marker in thebrain—close to 10% of administered dose. This is a high value by itself(80 fold higher than free drug), especially if this can also be achievedwith drugs of interest. These results indicate the high potential thisnovel DDS has for pathological conditions that require drug delivery tothe brain.

Run 2 comprised a mouse experiment. The animals used were healthyC57BL/6 mice. The DDS was LLG in nanoparticulate form. The marker wasMMC; the test system was the marker formulated in the novel DDS. Thedose administered was 5 mg/kg body weight, in both free and DDSformulations, 150 μl/animal. The DDS dose was 1 mg/ml.

The experiment was conducted with 4 animals, divided into two pairs. Onepair received the free marker, IN, into the right nostril. The otherpair received the marker/DDS formulation, IN, into the right nostril.Administration was slow, over several minutes, using an appropriatesyringe.

At 6 hours post administration, the animals were perfused through theheart, after which they were sacrificed. The brains were removed,homogenized, and the marker concentration was determined, as in Run 1.

The brains, post perfusion, were clean. The results obtained, expressedas % of administered dose, are shown in FIG. 17. The data are reportedper animal due to the animal-to-animal variability. Despite thevariability, the results are quite clear: negligible accumulation ofmarker occurred when it was administered in free form, and significantaccumulation when administered in the DDS form. As in the case of rats,the accumulation found when the marker was administered in the carrierconstitutes a positive finding in and of itself, and is 600-2,500 foldhigher than when the marker was administered in free form. These resultsshow that the potential of this drug delivery technology to deliverdrugs to the brain in a non-invasive route of administration is notlimited to a single animal species.

EXAMPLE 11 Animal Study Testing the Novel DDS in the Treatment ofDrug-Resistant Tumors in Mice: A Tumor Metastasis Model

The objective in the present study was to evaluate the novel DDS in atumor metastasis model. Similar to the previous study using mice and theinherent-MDR C-26 cell line, this study also involves an inherent MDRcell line, B16F10, from mouse melanoma. The specific protocolimplemented is established in the field and is designed to inducemetastasis in the lungs.

C57BL/6 female mice were used, which were 12 weeks old at initiation ofthe experiment. The tumor model was B16F10, cells injected i.v. Thechemotherapeutic drug employed was mitomycin C (MMC). The DDS system wasLLG in the form of nano particles. The test system was MMC formulated inthe novel DDS of the present invention, denoted MMC/DDS. The dose of MMCinjected was 5 mg/Kg of body weight and the DDS dose was 1 mg/ml.

The experiment was performed with 25 animals, divided into 5 groups,each group of 5 mice receiving a specific treatment as listed in Table10. Group 1 is a control group of healthy mice that were not inoculatedwith tumor cells.

TABLE 10 Animal Groups Group # 1 2 3 4 5 Treatment None Saline Free MMCFree DDS MMC/DDS

B16F10 cells were grown in cell culture flasks. At day zero, the cellswere harvested, washed several times, counted and immediately injectedto groups 2 to 5. The injected dose was 5×10⁵ cells in 50 μl PBS.

Treatments were given on days 1, 5 and 9. Administration was byinjection into the tail vein. All injected volumes were 0.1 ml. Theexperiment was terminated 21 days post tumor inoculation. The animalswere sacrificed, and the lungs were removed, weighed, and fixed inBouin's solution. Lung weight increase was calculated using thefollowing formula:

Lung weight increase (%)=100× (tumor lung weight−normal lungweight)/normal lung weight

Surface metastases were counted by an expert using a dissectingmicroscope. Sample codes were blinded so that the expert did not knowthe treatment each source animal received.

Quantitative evaluation of metastases in the lungs can be performed bytwo independent measurements: actual counting of the metastases inexcised and properly fixed lungs; and/or measurement of the increase inthe weight of the lungs due to the metastases in the animals injectedwith tumor cells. Both techniques were implemented in the present study.

The number of metastasis found in groups 1 to 5 are shown in FIG. 18.

As expected, there were no metastases in the lungs of the controlanimals that did not receive any tumor cells. All other groups thatreceived the i.v. injected B16F10 cells developed lung metastases. Themost aggressive metastatic situation developed in the animals thatreceived saline or free drug. As can be seen, there is no statisticaldifference between these groups, indicating that the inherent MDR natureof these cells is expressed in vivo also.

Treatment with the free DDS is seen to generate a 6 fold decrease in thenumber of metastases compared to saline, and treatment with the testformulation generated a much higher reduction, on the order of 17 fold.

In all four tumor-injected groups the weight of the lungs increasedcompared to that of normal animals (the control group). The resultsobtained are shown in FIG. 19.

The highest increase in lung weight—close to 400%—was seen in theanimals that did not receive any treatment (the saline group), and thegroup receiving treatment with free drug was almost the same. Theincrease in lung weight was smaller than no treatment and free drug forthe animals receiving the free DDS, but there was still a two-foldincrease in lung weight compared to control group of healthy animals.The best response—both in relative (compared to the other groups) and inabsolute (compared to healthy animals) terms—was observed with the testformulation of the MMC entrapped in the DDS. The % increase compared tohealthy animals was on the order of 10%, which is not statisticallysignificant, indicating the potential of this formulation to abolishlung metastasis.

Because the lung metastases are responsible for the increase in lungweight, there should be a reasonable correlation between the twoindependently measured parameters. This was the case, as clearly seen inFIG. 20, where the data (averages only) of FIGS. 18 and 19 werereplotted together. FIG. 20 also demonstrates the clearly superiorperformance of the test formulation in one of the most challenging tasksof tumor treatment, which is abolishing metastases from an MDR tumor.

To date, the performance of the novel DDS as a carrier forchemotherapeutic drugs in two independent animal models has beenstudied. One is a solid tumor and the other is lung metastases. In bothmodels, tumor cells injected into the animals, from the C-26 and B16F10cell lines were found to manifest in vivo their MDR nature previouslyseen in vitro.

In both models, treatment with the free DDS itself shows a betterclinical response than free drug. However, in both models the bestclinical response is seen with the test formulation of the novel DDSentrapping a chemotherapeutic drug. This indicates the high potentialfor this novel system in clinical use.

EXAMPLE 12 BSA-FITC Entry into MCF-7 Cells

Bovine serum albumin tagged with the fluorescent marker FITC (BSA-FITC)in both free form and entrapped in the DDS was used to determine whetherDDS can also induce the entry of large macromolecules into cells. Thefree and the DDS-entrapped BSA-FITC were incubated at 25° C. for 60minutes with confluent monolayers of MCF7 cells (originating from humanbreast carcinoma). MCF-7 cells are reported to have two known receptorsfor hyaluronic acid—ICAM-1 and CD44. The protein/DDS systems werecleaned from the free protein. The free and the entrapped protein wereat the same concentration: 3.3 mg/ml. At the end of the incubation thecells were viewed by means of confocal microscopy.

The results shown in the upper two panels of FIG. 21, are for freeprotein. Some of the protein gained entry into the cell, and can even beseen bound to the nuclear envelope, but not inside the nucleus. BSA isknown to bind non-specifically to cells, and may have gained entrythrough non-specific receptors or through pinocytosis.

The results in the lower two panels of FIG. 21 are for the DDS-entrappedBSA-FITC. Protein entry into the cells is considerably higher than forthe free protein, and the protein has also gained entry into thenucleus. As in the case of the entrapped EtBr (FIGS. 2-4), the exactmechanism by which this occurred is not yet fully understood. Thelikelihood that the protein-DDS is taken up by receptor-mediatedendocytosis is, however, even higher for the large protein than for thesmall EtBr.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and, therefore, such adoptions and modifications should and are intendedto be comprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

References

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Gottesman et al, “Genetic analysis of the multidrug transporter”, AnnuRev Genet 29:607-649 (1995)

Gref et al, “Biodegradable long-circulating polymeric nanospheres”,Science 263(5153):1600-1603 (1994)

Larsen et al, “Resistance mechanisms associated with alteredintracellular distribution of anticancer agents”, Pharmacol Ther85(3):217-29 (2000)

Margalit et al, J Controlled Release 17:285-296 (1991)

Nutt et al, “Differential expression of drug resistance genes andchemosensitivity in glial cell lineages correlate with differentialresponse of oligodendrogliomas and astrocytomas to chemotherapy”, CancerRes 60(17):4812-4818 (2000)

Van den Hoogen et al, “A microtiter plate assay for the determination ofuronic acids”, Anal Biochem 257(2):107-111 (1998)

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What is claimed is:
 1. A water-insoluble lipidated glycosaminoglycanparticle in the form of a sphere with the glycosaminoglycan portion ofthe particle forming a shell on the outside and the lipid portion of theparticle forming the inside, without the presence of liposome, saidparticle comprising the reaction product of at least oneglycosaminoglycan having a molecular weight within the range of about1×10⁵ to about 1×10⁷ daltons with at least one lipid having a primaryamine group, wherein the ratio of said lipid to said at least oneglycosaminoglycan is in the range of 1:1 or 5:1 to 20:1 w/w, saidparticle in the form of a sphere being capable of stably encapsulatingdrugs or other active ingredients that are soluble in water.
 2. Thewater-insoluble lipidated glycosaminoglycan particle of claim 1, whereinthe glycosaminoglycan is selected from the group consisting ofhyaluronic acid, keratan sulfate, chondroitin sulfate, heparin sulfate,heparan sulfate, dermatin sulfate, salts, and mixtures thereof.
 3. Thewater-insoluble lipidated glycosaminoglycan particle of claim 1, whereinthe glycosaminoglycan is hyaluronic acid.
 4. The water-insolublelipidated glycosaminoglycan particle of claim 1, wherein the lipid isphosphatidyl ethanolamine.
 5. The water-insoluble lipidatedglycosaminoglycan particle of claim 1, wherein the particle size rangesfrom about 2-5 microns.
 6. The water-insoluble lipidatedglycosaminoglycan particle of claim 1, wherein the particle size rangesfrom about 50-200 nanometers.
 7. The water-insoluble lipidatedglycosaminoglycan particle according to claim 1, wherein a water solubleactive ingredient is encapsulated within the particle.
 8. Thewater-insoluble lipidated glycosaminoglycan particle of claim 7, whereinthe water soluble active ingredient is selected from the groupconsisting of anti-infective agents, chemotherapeutic agents, proteins,hormones, enzymes, cells, and nucleic acids.
 9. The water-insolublelipidated glycosaminoglycan particle of claim 8, wherein the activeingredient is a chemotherapeutic agent for treating cancer.
 10. Thewater-insoluble lipidated glycosaminoglycan particle of claim 1encapsulating a marker used in imaging.
 11. The water-insolublelipidated glycosaminoglycan particle of claim 10, wherein the marker isa radioactive isotope.
 12. The water-insoluble lipidatedglycosaminoglycan particle according to claim 11, wherein theradioactive isotope is selected from the group consisting of ⁹⁹ _(Tc,)¹²⁵I and ⁶⁷Gd.
 13. The water-insoluble lipidated glycosaminoglycanparticle of claim 10, wherein the marker is a fluorescent molecule. 14.The water-insoluble lipidated glycosaminoglycan particle claim 7,wherein the water soluble active ingredient is a nucleic acid.
 15. Ascaffold for tissue engineering, comprising the water-insolublelipidated glycosaminoglycan particle of claim 1 encapsulating a wholecell.
 16. A method for preparing the water-insoluble lipidatedglycosaminoglycan particle of claim 1, comprising reacting at least oneglycosaminoglycan with at least one lipid having a primary amine group,wherein the ratio of said at least one lipid to said at least oneglycosaminoglycan is in the ratio of 1:1 or 5:1 to 20:1 w/w.
 17. Amethod for making the water-insoluble lipidated glycosaminoglycanparticle of claim 7 having a water soluble active ingredientencapsulated within, comprising reconstituting a lyophilized lipidatedglycosaminoglycan particle in water, and adding a powdered water solubleactive ingredient, whereby the water soluble active ingredient isencapsulated within the water-insoluble lipidated glycosaminoglycanparticle.
 18. In a method of diagnostic imaging, comprising the steps ofadministering a diagnostic agent to a patient and imaging said patient,the improvement wherein said diagnostic agent is the lipidatedglycosaminoglycan particle of claim
 10. 19. In a method of gene deliveryand short-term expression of nucleic acids for therapeutic purposescomprising administering to an animal in need thereof an effectiveamount of nucleic acids for therapeutic purposes, the improvementwherein said nucleic acids are formulated so as to be encapsulated inthe lipidated glycosaminoglycan particle of claim 1.